In vitro activity of cefiderocol against European Pseudomonas aeruginosa and Acinetobacter spp., including isolates resistant to meropenem and recent β-lactam/β-lactamase inhibitor combinations

ABSTRACT Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter spp. represent major threats and have few approved therapeutic options. Non-fermenting Gram-negative isolates were collected from hospitalized inpatients from 49 sites in 6 European countries between 01 January 2020 and 31 December 2020 and underwent susceptibility testing against cefiderocol and β-lactam/β-lactamase inhibitor combinations. Meropenem-resistant (MIC >8 mg/L), cefiderocol-susceptible isolates were analyzed by PCR, and cefiderocol-resistant isolates were analyzed by whole-genome sequencing to identify resistance mechanisms. Overall, 1,451 (950 P. aeruginosa; 501 Acinetobacter spp.) isolates were collected, commonly from the respiratory tract (42.0% and 39.3%, respectively). Cefiderocol susceptibility was higher than β-lactam/β-lactamase inhibitor combinations against P. aeruginosa (98.9% vs 83.3%–91.4%), and P. aeruginosa resistant to meropenem (n = 139; 97.8% vs 12.2%–59.7%), β-lactam/β-lactamase inhibitor combinations (93.6%–98.1% vs 10.7%–71.8%), and both meropenem and ceftazidime-avibactam (96.7% vs 5.0%–45.0%) or ceftolozane-tazobactam (98.4% vs 8.1%–54.8%), respectively. Cefiderocol and sulbactam-durlobactam susceptibilities were high against Acinetobacter spp. (92.4% and 97.0%) and meropenem-resistant Acinetobacter spp. (n = 227; 85.0% and 93.8%) but lower against sulbactam-durlobactam- (n = 15; 13.3%) and cefiderocol- (n = 38; 65.8%) resistant isolates, respectively. Among meropenem-resistant P. aeruginosa and Acinetobacter spp., the most common β-lactamase genes were metallo-β-lactamases [30/139; blaVIM-2 (15/139)] and oxacillinases [215/227; blaOXA-23 (194/227)], respectively. Acquired β-lactamase genes were identified in 1/10 and 32/38 of cefiderocol-resistant P. aeruginosa and Acinetobacter spp., and pirA-like or piuA mutations in 10/10 and 37/38, respectively. Conclusion: cefiderocol susceptibility was high against P. aeruginosa and Acinetobacter spp., including meropenem-resistant isolates and those resistant to recent β-lactam/β-lactamase inhibitor combinations common in first-line treatment of European non-fermenters. IMPORTANCE This was the first study in which the in vitro activity of cefiderocol and non-licensed β-lactam/β-lactamase inhibitor combinations were directly compared against Pseudomonas aeruginosa and Acinetobacter spp., including meropenem- and β-lactam/β-lactamase inhibitor combination-resistant isolates. A notably large number of European isolates were collected. Meropenem resistance was defined according to the MIC breakpoint for high-dose meropenem, ensuring that data reflect antibiotic activity against isolates that would remain meropenem resistant in the clinic. Cefiderocol susceptibility was high against non-fermenters, and there was no apparent cross resistance between cefiderocol and β-lactam/β-lactamase inhibitor combinations, with the exception of sulbactam-durlobactam. These results provide insights into therapeutic options for infections due to resistant P. aeruginosa and Acinetobacter spp. and indicate how early susceptibility testing of cefiderocol in parallel with β-lactam/β-lactamase inhibitor combinations will allow clinicians to choose the effective treatment(s) from all available options. This is particularly important as current treatment options against non-fermenters are limited.

A ntimicrobial resistance is widespread throughout Europe (1).In particular, the high rates of carbapenem resistance observed in Pseudomonas aeruginosa and Acinetobacter spp.(19% and 48%, respectively, in 2021) represent a major threat, considering few approved therapeutic options are available (1,2).The World Health Organization has, therefore, recognized carbapenem-resistant (CR) P. aeruginosa and CR Acinetobacter baumannii (CRAB) as critical priority pathogens (2).
Cefiderocol is a unique catechol-siderophore cephalosporin approved in Europe for the treatment of infections due to aerobic Gram-negative organisms in adults with limited treatment options (15).The mechanism of action of cefiderocol is the disruption of peptidoglycan cell-wall synthesis via inhibition of penicillin-binding proteins (PBPs) (16,17).The structure of cefiderocol and "Trojan Horse" mechanism of bacterial cell entry provide enhanced stability to a wide range of β-lactamases, and allow for the activity of cefiderocol to be broadly unaffected by efflux pump overexpression and porin channel modifications observed in CR non-fermenters (17)(18)(19)(20)(21).
There is notable variation in the European Society of Clinical Microbiology and Infectious Diseases and Infectious Diseases Society of America guidance for treatment of infections due to CR P. aeruginosa and Acinetobacter spp., although both gener ally recognize that in vitro activity of antimicrobials is an important consideration for treatment decision-making (33,34).The longitudinal surveillance studies SENTRY and SIDERO generated data on the susceptibilities of cefiderocol and approved BLBLI combinations against non-fermenters, including CR and BLBLI combination-resistant isolates (35,36).Although, despite the current and anticipated use of cefiderocol and BLBLI combinations against CR Gram-negative infections, there remains limited data comparing these antimicrobials and developmental BLBLI combinations against CR non-fermenter isolates, including those also resistant to a comparator antimicrobial.
The aim of this study was to evaluate the in vitro activity of cefiderocol, merope nem, BLBLI combinations (approved and in development), and colistin against clinical Gram-negative isolates collected between 01 January 2020 and 31 December 2020, across six countries in Europe.Here, we report the results for P. aeruginosa and Acinetobacter spp.isolates.Results for Enterobacterales isolates collected in this study are reported elsewhere.

Epidemiology
In total, 1,451 isolates were collected from European hospitals, of which 950 (65.5%) were P. aeruginosa and 501 (34.5%) were Acinetobacter spp.[including 458 (91.4%) A. baumannii complex] (see Table S1 for isolates by country).P. aeruginosa and Acinetobacter spp.isolates were collected from a range of infection sources, the most common for both species being respiratory tract [42

Susceptibility profiles of isolates
Susceptibility to cefiderocol was 98.9% against P. aeruginosa isolates and 92.4% against Acinetobacter spp.isolates [Table 1; see Table S2 for susceptibility rates using Clini cal and Laboratory Standards Institute (CLSI) breakpoints or United States Food and Drug Administration (FDA) breakpoints where CLSI breakpoints were not available].By individual country, susceptibility to cefiderocol was similar against P. aeruginosa, ranging from 98.7% in France to 99.3% in the United Kingdom, but showed more variability against Acinetobacter spp.(88.7% in Italy to 98.0% in Spain) (Table S3A through E).Against P. aeruginosa isolates, rates of susceptibility to BLBLI combinations were lower than cefiderocol, ranging from 83.3% for imipenem-relebactam to 91.4% for cefepime-taniborbactam (Table 1).Against Acinetobacter spp.isolates, rates of suscepti bility were 92.4% to cefiderocol and 97.0% to sulbactam-durlobactam (FDA breakpoint) (Table 1) (37,38).Using epidemiological cut-off (ECOFF) values, susceptibility to colistin was 99.7% and 98.4% for P. aeruginosa and Acinetobacter spp., respectively (Table 1).
Susceptibility to cefiderocol remained high against P. aeruginosa isolates resistant to BLBLI combinations, ranging from 93.6% against ceftazidime-avibactam-resistant isolates to 98.1% against imipenem-relebactam-resistant isolates (Table 2).On the other hand, BLBLI combinations did not show high levels of activity against BLBLI combinationresistant isolates, with the highest susceptibility being 71.8% for ceftolozane-tazobactam against aztreonam-avibactam-resistant isolates.Colistin showed 100% susceptibility against BLBLI combination-resistant phenotypes.The activity of antimicrobials against cefiderocol-resistant P. aeruginosa was not analyzed due to the low number of isolates (n = 10).Against P. aeruginosa isolates that were resistant to both meropenem and ceftazidime-avibactam, susceptibility to cefiderocol was 96.7%, which was higher than any BLBLI combination (≤45.0%susceptibility) (Table 2).Similarly, against meropenemand ceftolozane-tazobactam-resistant isolates, susceptibility to cefiderocol was higher than all tested BLBLI combinations (98.4% vs <55% susceptibility, respectively).Suscepti bility rates for antibiotics against antibiotic-resistant P. aeruginosa using CLSI breakpoints are reported in Table S2.

β-Lactamase genes in meropenem-resistant pathogens
Of the 139 meropenem-resistant P. aeruginosa isolates, 2.9% (4/139) were cefiderocol resistant and were analyzed by whole-genome sequencing (WGS), while the remain ing isolates were analyzed by PCR for the presence of specific β-lactamase genes.Antimicrobial susceptibility rates in meropenem-resistant P. aeruginosa according to β-lactamase genes identified are reported in Table S5A.The most common β-lactamase b Antimicrobials were tested against P. aeruginosa and/or Acinetobacter spp.based on expected use in a real-world setting.Results are not reported for isolates tested against antibiotics to which they had an expected resistance phenotype.Susceptibility was assessed according to EUCAST breakpoints (including non-species-specific PK/PD breakpoints, high dosage breakpoints, and breakpoints for the agent without inhibitor, where applicable), except for sulbactam-durlobactam and colistin where FDA breakpoints and ECOFF values were used, respectively.Data are shown where n ≥ 20 isolates were available.Data on susceptibility to colistin are shown in parentheses as colistin is not recommended for monotherapy and is not associated with a clinical monotherapy breakpoint (as per EUCAST v.14.0 guidance).c Refers to susceptibility, or susceptibility with increased exposure for meropenem, meropenem-vaborbactam, aztreonam-avibactam, and cefepime-taniborbactam. d Includes 458 A. baumannii complex isolates.e Sulbactam-durlobactam-resistant isolates were included irrespective of the number of isolates due to the scarcity of published data.

β-Lactamase genes and other potential resistance mechanisms identified in cefiderocol-resistant pathogens
In total, 10 P. aeruginosa and 38 Acinetobacter spp.(37 A. baumannii; 1 Acinetobacter calcoaceticus) isolates were cefiderocol resistant and were analyzed by WGS.
Half (19/38) of the cefiderocol-resistant Acinetobacter spp.isolates were found to be ST2, collected in all six European countries, while the other half consisted of isolates of other STs, including two novel STs from France and Germany (

DISCUSSION
This study provides additional data on the in vitro susceptibilities of cefiderocol and BLBLI combinations, including those still in development, against a large collection of European isolates of glucose non-fermenting Gram-negative bacteria.The data collected in this study are from a greater number of sites per European country compared with the longitudinal SENTRY and SIDERO surveillance programs, which are more geographically spread (35,36).The susceptibility rate for cefiderocol was higher than BLBLI combinations (including those still in development, such as aztreonam-avibactam and cefepime-taniborbactam) against P. aeruginosa overall (98.9% vs 83.3%-91.4%,respectively) and meropenemresistant isolates (97.8% vs ≤59.7%).These observations are consistent with similar previous in vitro studies (5,26,(39)(40)(41)(42)(43)(44)(45).It is important to note that meropenemresistant P. aeruginosa isolates in this study were defined according to the EUCAST MIC resistance breakpoint for high-dose (2 g), extended (3-h)-infusion meropenem (>8 mg/L), to represent isolates that are meropenem resistant even when treated with the highest meropenem dose available to patients.As some previous studies have defined meropenem-resistant/non-susceptible P. aeruginosa according to the EUCAST MIC resistance breakpoint for standard-dose meropenem (>2 mg/L), susceptibility rates for BLBLI combinations tested in previous studies may be higher (39,43).
Of the β-lactamase genes observed in meropenem-resistant P. aeruginosa isolates, most were MBLs (most commonly VIM), against which cefiderocol retains high activ ity, in contrast to most BLBLI combinations (46).Low frequencies (≤5%) of IMP, NDM (bla NDM-1 ), and GES were observed, lower than previously published data (6).The 71.2% of meropenem-resistant P. aeruginosa isolates which did not harbor β-lactamase genes of interest likely exhibited non-β-lactamase mechanisms of resistance, such as increased expression of efflux systems, chromosomal cephalosporinase activity, or reduced porin expression (3)(4)(5)(6)47).Susceptibility to cefiderocol was higher than to BLBLI combinations against BLBLI combination-resistant P. aeruginosa (93.6%-98.1% vs 12.2%-71.8%,respectively).Similarly, susceptibility to cefiderocol was high against meropenem-resistant P. aeruginosa resistant to ceftazidime-avibactam or ceftolozane-tazobactam (≥96.7%), while ceftolozanetazobactam and ceftazidime-avibactam both had poorer activity (≤30.6%).This is indicative of a low degree of cross resistance between cefiderocol and BLBLI combinations and a particularly high degree of cross resistance between ceftolozane-tazobactam and ceftazidime-avibactam.Previous studies have also shown cefiderocol to have much higher activity than ceftazidime-avibactam or ceftolozane-tazobactam against ceftazidimeavibactam-or ceftolozane-tazobactam-resistant P. aeruginosa (6,48,49).Given that 11%-23% of P. aeruginosa isolates show resistance to ceftazidime-avibactam or ceftolozanetazobactam, cefiderocol would be the preferred agent over BLBLI combinations for treatment of infections caused by such isolates.Even aztreonam-avibactam and cefepimetaniborbactam, which are still in development, showed lower susceptibility (≤54.8%)compared with cefiderocol (≥96.7%)against meropenem-resistant P. aeruginosa resistant to ceftazidime-avibactam or ceftolozane-tazobactam.Lower activity of cefepime-tanibor bactam was previously demonstrated against ceftolozane-tazobactam-and ceftazidimeavibactam-resistant/-non-susceptible P. aeruginosa, as taniborbactam does not fully restore cefepime activity in some BLBLI combination-resistant P. aeruginosa isolates, such as IMP producers (Table S5) (39,50).Further, studies on the in vitro activity of aztreonamavibactam showed poor activity against P. aeruginosa overall (44,45).Cefiderocol-resistant P. aeruginosa isolates were collected across Europe in this study.Although isolates were not collected under a surveillance program, the diverse range of STs indicates that cefiderocol resistance in P. aeruginosa arises from specific clones and is not due to clonal expansion.Although there were no clear patterns of resist ance mechanisms in cefiderocol-resistant P. aeruginosa isolates in this study, previous observations have noted the common presence of mutations in genes encoding the PiuA and PirA receptors required for the uptake of siderophore conjugates (51)(52)(53)(54) and the role of mutations in the ftsI gene encoding PBP3 (50).Further investigations are required to confirm whether mutations in piuA, pirA, and ftsI impact drug resistance or are natural polymorphisms.
Acinetobacter spp.are particularly difficult to treat due to the prevalence of antimi crobial resistance (2).However, both cefiderocol and sulbactam-durlobactam demon strated good in vitro activity against Acinetobacter spp.overall (>90% susceptibility) and meropenem-resistant isolates (85.0% and 93.8% susceptibility, respectively), in agreement with previous studies (5,55,56).Against Acinetobacter spp.overall, cefiderocol demonstrated a lower MIC 90 than sulbactam-durlobactam.To the authors' knowledge, this is the first published study directly comparing the in vitro activity of cefiderocol and sulbactam-durlobactam, which was recently approved by the United States Food and Drug Administration for the treatment of hospital-acquired/ventilatorassociated bacterial pneumonia caused by susceptible A. baumannii complex in adults (57,58).While susceptibility to sulbactam-durlobactam was higher than to cefiderocol against meropenem-resistant Acinetobacter spp., the FDA breakpoint for sulbactam-dur lobactam (≤4 mg/L) was used, as EUCAST breakpoints are not available (37).Had the CLSI breakpoint been used in place of the EUCAST non-species-specific PK/PD breakpoint for cefiderocol (≤4 mg/L vs ≤2 mg/L), cefiderocol susceptibility against meropenem-resistant Acinetobacter spp.would be more comparable to sulbactam-durlobactam (88.5%) (Table S2) (59).
Cefiderocol-resistant Acinetobacter spp.accounted for 7.6% of Acinetobacter spp.collected in this study, against which sulbactam-durlobactam demonstrated good in vitro activity.Although some cefiderocol-resistant isolates had similar genotypic and phenotypic data, which may suggest clonality, isolates were not collected under a surveillance program and numbers were low.Mutations in piuA were found in 97.4% of isolates, suggesting that piuA may be the major iron-regulated outer membrane protein involved in the uptake of cefiderocol in Acinetobacter spp.Half of all cefiderocol-resistant Acinetobacter spp.isolates had mutations in the ftsI gene encoding PBP3, which cefiderocol is known to inhibit (52).The retained activity of sulbactam-durlobactam against cefiderocol-resistant Acinetobacter spp.suggests that there does not appear to be any cross resistance due to PBP3 target-site mutations.This was unexpected, as sulbactam also inhibits PBP3, and sulbactam-durlobactam resistance has previously been attributed to PBP3 mutations (32,63); in addition, Acinetobacter spp.resistant to sulbactam-durlobactam (3.0% of isolates) also had low susceptibility to cefiderocol (13.3%).It may be a concern that sulbactam-durlobactam resistance is being observed in Europe this early in the use of this treatment, possibly as a consequence of prior exposure to ampicillin-sulbactam.However, this study was not designed to comprehensively investigate mechanisms of resistance in non-fermenter isolates.
The data of this study do provide insights into the therapeutic options for infections due to P. aeruginosa and Acinetobacter spp. with resistant phenotypes.The low levels of cross resistance observed between cefiderocol and any BLBLI combination, with the exception of sulbactam-durlobactam, support the concept that cefiderocol should be tested at the same time as these BLBLI combinations to allow clinicians to choose effective treatment(s) for non-fermenter infections from all available options.It is particu larly important that all effective treatment(s) are identified and considered, as current treatment options are limited (33,34).Importantly, the high cross resistance observed between ceftazidime-avibactam and ceftolozane-tazobactam in this and other studies suggests that cycling between these treatments to treat infections due to P. aeruginosa is unlikely to be an appropriate option (64)(65)(66).The susceptibilities of aztreonam-avibactam and cefepime-taniborbactam against P. aeruginosa resistant to meropenem and both meropenem and BLBLI combinations were low, which also suggests that these will not be good treatment options for infections due to P. aeruginosa.
Although susceptibility to colistin was high against meropenem-resistant P. aeruginosa and Acinetobacter spp.isolates, colistin is known to have concerningly high rates of nephrotoxicity and poor tissue penetration, particularly in the lungs (67)(68)(69), and the majority of non-fermenter isolates in this study were from respiratory tract infections.Colistin is also not recommended by EUCAST for monotherapy and is not associated with a clinical monotherapy breakpoint (70).
There are several limitations to this study.Isolates were only collected from six European countries, and up to 35 non-fermenter isolates per participating site were included.Hence, there were low numbers of some isolates resistant to at least one antimicrobial, particularly cefiderocol.Ceftazidime-avibactam was tested using a validated commercial method, while other antimicrobials were tested using custom plates.A selection of potential mechanisms of resistance was only screened for in meropenem-and cefiderocol-resistant isolates, using different methodologies and screening panels, and excluding analysis of expression levels of genes or other potential mechanisms of BLBLI combination resistance, such as PBP1 and PBP2.Therefore, robust interpretations of resistance mechanisms and any cross resistance could not be made.The STs of cefiderocol-susceptible isolates were not identified, so clonal expansion of isolates with resistant phenotypes was not determined; however, these non-surveillance data would not have accurately reflected clonal epidemiology in Europe.Lastly, in vitro data cannot replace clinical studies in patients, and in vitro activity may not reflect in vivo efficacy of a therapy in clinical practice.

Conclusions
These results confirm the high levels of in vitro activity of cefiderocol against Gram-negative P. aeruginosa and Acinetobacter spp.isolates from Europe, including both meropenem-resistant isolates and those resistant to recent BLBLI combina tions commonly used in first-line treatment of CR infections.Cefiderocol often had high in vitro activity where the majority of BLBLI combinations did not, and there was no apparent cross resistance between cefiderocol and BLBLI combinations, with the exception of sulbactam-durlobactam.

Clinical isolates
Between 01 January and 31 December 2020, Gram-negative clinical isolates from hospitalized inpatients were collected at 49 sites across Austria, France, Germany, Italy, Spain, and the United Kingdom (see Table S7 for details of participating centers).Each site was requested to collect 20 P. aeruginosa and 15 A. baumannii (Enterobacterales isolates were also collected as part of the overall study, for which the methods and results are reported elsewhere).Isolates included those from all infection sources, with the exception of the urinary tract.Only one isolate of the same genus and species was allowed per patient.Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry was used for species identification at International Health Management Associates (IHMA) Europe Sàrl (Monthey, Switzerland).
International Organization for Standardization 20776-1 susceptibility testing standards and EUCAST guidance were followed for the preparation of antimicrobials for testing and MIC determinations (71,72); tryptic soy agar plates containing 5% sheep blood were sourced from Liofilchem (Roseto degli Abruzzi, Italy; product code: 11037), cation-adjusted Mueller-Hinton broth was sourced from Becton Dickinson (Franklin Lakes, NJ, USA; product code: 212322), and iron-depleted cation-adjusted Mueller-Hinton broth (used for cefiderocol testing) was prepared by IHMA Europe Sàrl.This excludes ceftazidime-avibactam, for which MIC values were only available when Sensititre freeze-dried panels (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used in the preparation of ceftazidime-avibactam for testing.All antibiotics were tested daily using the quality control strains P. aeruginosa ATCC 27853 (as recommended by EUCAST and CLSI, and in line with guidance from CLSI) and A. baumannii ATCC 13304 (in line with guidance from CLSI, where a same-species quality control strain was not recommended by EUCAST) (59,73).The MIC values for each tested antibiotic were manually read as the lowest concentration inhibiting visible growth.For cefiderocol and meropenem, MIC values were determined more than once; a third MIC determination was carried out if MIC values differed by >1 dilution, and the geometric mean was reported.
Meropenem resistance was defined using a breakpoint of MIC >8 mg/L, relating to high-dose, extended-infusion (2 g, 3-h infusion) meropenem; similarly, isolates with a meropenem MIC >8 mg/L when tested with a fixed vaborbactam concentration of 8 mg/L were considered resistant to meropenem-vaborbactam.Aztreonam-avibactam, cefepime-taniborbactam, and sulbactam-durlobactam do not currently have approved EUCAST MIC breakpoints; nor does cefiderocol for Acinetobacter spp.For aztreonam-avi bactam and cefepime-taniborbactam, EUCAST breakpoints for high-dose aztreonam and high-dose cefepime alone were used.For Acinetobacter spp., the non-species-specific PK/PD breakpoint (37) was used for cefiderocol and the FDA breakpoint (38) was used for sulbactam-durlobactam.For colistin, EUCAST ECOFF values were used.

Identification of β-lactamase genes in meropenem-resistant isolates
Isolates with a meropenem MIC >8 mg/L and a cefiderocol MIC ≤2 mg/L were analyzed by PCR (performed by IHMA Europe Sàrl) to identify the presence of β-lactamase genes that may confer meropenem resistance (see Table S10 for genes and primers used).Data on β-lactamase genes in isolates that were meropenem resistant (meropenem MIC >8 mg/L) and cefiderocol resistant (MIC >2 mg/L) were generated by WGS (see below).
DNA extraction was performed from a single colony obtained from a fresh tryptic soy blood agar culture for each isolate, using the QIAGEN TissueLyser II instrument (Hilden, Germany) as per manufacturer instructions.Preparations then underwent PCR amplification and sequencing to screen for the presence of genes encoding clinically relevant β-lactamases: ESBLs (bla SHV , bla TEM , bla CTX-M , bla VEB , bla PER , bla GES ), AmpCs [bla ACC , bla CMY I/MOX , bla CMY II , bla DHA , bla FOX , bla ACT-MIR , bla PDC (in P. aeruginosa only)], and carbapenemases (bla KPC , bla OXA , bla NDM , bla IMP , bla VIM , bla SPM , bla GIM , bla GES ).Amplicons were sequenced by Fasteris (Geneva, Switzerland) and then analyzed using SeqScape Software 3 (Thermo Fisher Scientific Inc.; Waltham, MA, USA).Limited sequencing was used to screen bla TEM and bla SHV to identify TEM-type and SHV-type enzymes containing amino acid substitutions common to ESBLs (bla TEM : amino acid 104, 164, 238, 240; bla SHV : amino acid 146, 179, 238, 240) and to screen bla CTX-M (groups 1, 2, 8, 9, and 25) to identify CTX-M-type enzymes containing the D240G amino acid substitution associated with elevated ceftazidime MIC values.Genes encoding SHV-type and TEM-type enzymes were reported as ESBL or original-spectrum β-lactamase genes.The 16S ribosomal DNA for all isolates was also amplified by PCR and sequenced for bacterial identification.

Identification of β-lactamase genes and other potential resistance mecha nisms in cefiderocol-resistant isolates
Isolates with a cefiderocol MIC >2 mg/L were analyzed by WGS to identify possible mechanisms of resistance.DNA isolation was performed using the QIAGEN QIAamp DNA Mini kit, and library preparation was performed using the Illumina DNA Prep kit (San Diego, CA, USA) at IHMA, Inc. (Schaumburg, IL, USA).Libraries were then shipped to Azenta (South Plainfield, NJ, USA), where short-read WGS (2 × 150 base pairs; paired-end) was performed on an Illumina HiSeq platform to a 100× depth of coverage.Quality control was performed using the CheckM lineage workflow (74)(75)(76) to assure low contamination (≤5%) and completeness of assemblies (≥95%) were achieved.The multilocus sequence typing scheme Pasteur was used to determine relatedness of Acinetobacter spp.isolates.
Genomic assemblies were created using the QIAGEN CLC Genomics workbench (v.21.0.5).In order to identify β-lactamase genes of interest, assemblies were queried using the ResFinder database (77) with coverage and identity thresholds of ≥35% and ≥72%, respectively.Genes identified with <100% identity or coverage were evaluated for a variant by pairwise alignment to a reference sequence using the ResFinder database (77).Variants were defined using the Bacterial Antimicrobial Resistance Reference Gene Database from the National Center for Biotechnology Information (Bioproject 313047).
Non-β-lactamase genes of interest in this study included those encoding PBP3 (ftsI), porins [oprD (P.aeruginosa) and carO (Acinetobacter spp.)], and those related to iron acquisition [pirA-like, piuA, piuC, and pvdS (P.aeruginosa)].Genes were analyzed by pairwise alignment and classified as wild type if they had 100% amino acid sequence identity to the species-specific reference sequence (Table S11).These genes were also screened for gross disruption vs species-specific reference sequences (Table S11) and were considered to have gross disruption if the coding sequence carried a nonsense mutation, frameshift, indels of >20 codons, or ablation of the canonical start or stop codons without a replacement immediately adjacent and in-frame.Genes were not considered disrupted if there were ablated start or stop codons immediately adjacent to intact, in-frame start or stop codons.Genes were listed to be not found if a BLAST search with the reference gene yielded no hit with E-value <1E−25.

TABLE 1
In vitro activity of cefiderocol, BLBLI combinations, and other relevant antibiotics against P. aeruginosa and Acinetobacter spp.isolates a,b Antibiotics were tested against P. aeruginosa and/or Acinetobacter spp.based on expected use in a real-world setting.Susceptibility was assessed according to EUCAST breakpoints (including non-species-specific PK/PD breakpoints, high-dosage breakpoints, and breakpoints for the agent without inhibitor, where applicable), except for sulbactam-durlobactam and colistin where FDA breakpoints and ECOFF values were used, respectively.Data on susceptibility to colistin are shown in parentheses as colistin is not recommended for monotherapy and is not associated with a clinical monotherapy breakpoint (as per EUCAST v.14.0 guidance).

TABLE 5 β
-Lactamase genes and other potential resistance mechanisms identified in cefiderocol-resistant P. aeruginosa isolates (n = 10) a,b a FDC, cefiderocol; MBL, metallo-β-lactamase; NDM, New Delhi MBL; OXA, oxacillinase; PDC, Pseudomonas-derived cephalosporinase; ST, sequence type; UK, United Kingdom; WT, wild type.b Data were generated by whole-genome sequencing.A gene was considered to have a gross disruption if the coding sequence carried a nonsense mutation, frameshift, indels of >20 codons, or ablation of the canonical stop or start codons without a replacement immediately adjacent and in-frame.Genes were listed to be not found if a BLAST search with the reference gene yielded no hit with E-value <1E-25.Non-β-lactamase genes that were either found to have gross disruptions or mutations or were not found are shown in gray.c Data shown are a curated summary.d Indicates a novel ST.

TABLE 6 β
-Lactamase genes and other potential resistance mechanisms identified in cefiderocol-resistant Acinetobacter spp. a isolates (n = 38) b,c (Continued on next page)

TABLE 6 β
-Lactamase genes and other potential resistance mechanisms identified in cefiderocol-resistant Acinetobacter spp. a isolates (n = 38) b,c (Continued) Data were generated by whole-genome sequencing.A gene was considered to have a gross disruption if the coding sequence carried a nonsense mutation, frameshift, indels of >20 codons, or ablation of the canonical stop or start codons without a replacement immediately adjacent and in-frame.Genes were listed to be not found if a BLAST search with the reference gene yielded no hit with E-value <1E-25.Non-β-lactamase genes that were either found to have gross disruptions or mutations or were not found are shown in gray.